Detecting material state changes in the nucleolus by label-free digital holographic microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Ribosome biogenesis occurs in the nucleolus, a dynamic biomolecular condensate.
• Nucleolar morphology serves as a biomarker for cellular stress and disease.
• Liquid-liquid phase separation drives the formation of the nucleolus.

Purpose of the Study:

• To develop a label-free method for detecting and quantifying nucleoli using digital holographic microscopy (DHM).
• To assess the material state of nucleoli and other cellular condensates.
• To explore DHM's potential for identifying pathological condensates and cellular structures.

Main Methods:

• Digital holographic microscopy (DHM) was employed for label-free imaging of human cells.
• Convolutional neural networks were trained for automated detection and quantification of nucleoli.
• A novel index based on optical thickness was developed to characterize nucleoli.

Main Results:

• DHM successfully detected and quantified nucleoli in both adherent and suspension human cells.
• The developed index distinguished nucleoli with modulated material states (optogenetics, drug treatment, protein depletion).
• DHM showed potential in detecting other condensates like those from mutant huntingtin, ataxin-3, TDP-43, and lipid droplets.

Conclusions:

• DHM is a powerful, label-free tool for quantitative characterization of nucleoli.
• The technology can assess the material state of nucleoli and other cellular assemblies.
• DHM offers a non-invasive approach for studying cellular condensates and their functions.